DE69534354T2 - CHIMERIC cDNA CLONE OF THE VIRUS OF INFECTIOUS BURSAL DISEASE, EXPRESSION PRODUCTS AND BASED VACCINES - Google Patents

Abstract

Chimeric cDNA for the expression of immunogenic polypeptides include the genetic epitopic determinants for a base infectious bursal disease virus strain and at least one other infectious bursal disease virus strain. The genetic epitopic determinants encode amino acids or amino acid sequences which define epitopes bound to by previously established monoclonal antibodies. The immunogens expressed by the cDNA may be employed to provide a vaccine against a plurality of IBDV strains. The epitopic determinant of IBDV lethal strains has been detected, and an immunogen for conferring immunity with respect thereto is disclosed. Similarly, a monoclonal antibody specific for IBDV lethal strains is identified, and a vaccine for passive immunization therewith is also disclosed. Immunogens exhibiting conformational epitopes, in the form of virus-like particles, are effective in the preparation of vaccines.

Description

technical area

The The present invention provides chimeric IBDV immunogens which are active against virulent and lethal challenge by classic IBDV strains and protect their variants, and methods of obtaining vaccines containing these chimeric immunogens and vaccines.

background and state of the art

The infectious Bursitis virus (IBDV) is for a highly contagious immunosuppressive disease in young chickens, which causes significant losses for the poultry industry worldwide (In retrospect summarized in Kibenge (1988) J. Gen. Virol. 69: 1757-1775). Infection of susceptible chickens with virulent IBDV strains can lead to a highly contagious immunosuppressive condition that as infectious Bursitis (IBD) is known. Damage to the lymphoid follicles of the Inflicted by Fabricius and the spleen may facilitate infections, those inflicted by other means be, and can be the ability of a chicken, to respond to vaccinations (Cosgrove (1962) "Avian Dis.", 6: 385-3894).

It There are two serotypes of IBDV (McFerran et al. (1980) Avian Path., 9: 395-404). Serotype 1 viruses pathogenic for Chicken and differ significantly in their virulence (Winterfield et al. (1978) "Avian Dis.", 5: 253-260), whereas Serotype 2 viruses derived from turkeys were isolated for Chicken are avirulent (Ismail et al., (1988) Avian Dis., 32: 757-759, Kibenge (1991) Virology, 184: 437-440).

IBDV is a member of the Birnaviridae family, and whose genome exists from two segments double-stranded RNA (Dobos et al., (1979) J. Med. Virol. ", 32: 593-605) smaller segment B (-2800 bp) codes for VP1, the dsRNA polymerase, The bigger genomic Segment A (-3000 bp) codes for a 110 kDa precursor polyprotein in a single open reading frame (ORF) leading to mature VP2, VP3 and VP4 (Azad et al. (1985) Virology, 143: 35-44). Out a small ORF partially overlapping the polyprotein ORF, Segment A may be the same for Encode VP5, a 17 kDa protein of unknown function (Kibenge et al. (1991) "J. Gene. Virol. ", 71: 569-577).

During VP2 and VP3 are the major structural proteins of the virion, VP2 the main host-protective Immunogen and causes the induction of neutralizing antibodies (Becht et al. (1988) "J. Gene. Virol. ", 69: 631-640; Fahey et al. (1989) "J. Gene. Virol. ", 70: 1473 to 1481). VP3 is considered to be a group-specific antigen as it passes through monoclonal antibodies (Mabs) against VP3 from both strains of both serotype 1 and also serotype 2) (Becht et al. (1988) J. Gen. Virol., 69: 631-640). VP4 is a virus-encoded protease and is involved in the processing of Precursor protein involved (Jagadish et al., (1988) J. Med. Virol., 62: 1084-1087).

In The past has been the control of IBDV infection in young chickens Live vaccination with avirulent strains achieved or essentially by the transfer of a maternal Antibody induced by administering live and killed IBDV vaccines to brood hens. Unfortunately have been in recent years virulent variant strains of IBDV isolated from vaccinated flock of birds in the United States (Snyder et al., 1988b) "Avian Dis ", 32: 535-539; Van Marel et al. (1990) "Dsch. Tierarztl. Wschr. ", 97: 81-83) Use of a selected one Offer to Mabs raised against various IBDV strains to the identification of course occurring GLS, DS326, RS593 and Delaware variant viruses in the United States. Considerable economic Losses are due to the appearance of these antigenic variants (Delaware and GLS) in the field (Snyder et al., (1992) "Arch. Virol.", 127: 89-101).

These variant strains are antigenically different from the classical IBDV strains, which typically isolated before 1985 and lack an epitope or epitopes, defined by neutralizing monoclonal antibodies (Mabs) B69 and R63 (Snyder et al. (1988a) "Avian Dis.", 32: 527-534, Snyder et al. (1988b) "Avian Dis.", 32: 535-539; Snyder et al. (1992) "Arch. Virol. "127: 89-101) The occurrence of these variant strains in the field has become many commercially available living and killed Vaccines for IBDV reformulated in an attempt to cope with the larger antigenic spectrum of Viruses that have been identified as circulating in the field, correspond to.

Attempts to develop a recombinant vaccine for IBDV were performed, and the genome of IBDV has been cloned (Azad et al. (1985) Virology, 143: 35-44). The VP2 gene of IBDV has been cloned and expressed in yeast (Macreadie et al (1990) "Vaccine", 8: 549-552) as well as in a recombinant avipox virus (Bayliss et al. (1991) "Arch. Virol." , 120: 193-205). When chickens were immunized with the VP2 antigen expressed in yeast, the antisera gave passive protection in chickens against IBDV infection. When used in active immunization studies, the fowlpox virus-transmitted VP2 antigen afforded protection against mortality, but not against Fabricius' bursa (bursa) damage.

Recently became the synthesis of VP2, VP3 and VP4 structural proteins of the variant GLS-IBDV strain in a baculovirus expression system (Vakharia et al., (1993) J. Med. Gene. Virol., 74: 1201-1206). An initial two-dose active immunity study in SPF chickens was baculovirus-expressed GLS proteins capable of providing 79% protection against a virulent GLS challenge to confer (Vakharia et al., (1993) J. Gen. Virol., 74: 1201-1206). In a subsequent extended Study of active crossimmunity by raising the antigenic mass of baculovirus-expressed GLS protein a complete one Protection against the variant GLS and E / Del strains with a single dose received, but only a partial protection was against the classic STC strain obtained unless two doses were administered.

In In recent years, the large segment nucleic acid sequences have become A of five Serotype 1 IBDV strains; 002-73 (Hudson et al. (1986) Nucleic Acids Res. ", 14: 001-5012), Cu-1, PBG98, 52/70 (Bayliss et al. (1990) J. Gen. Virol., 71: 1303-1312), STC (Kibenge (1990) J. Gen. Virol., 71: 569-577) and serotype 2 OH strain (Kibenge (1991) Virology, 184: 437-440). additionally was the VP2 gene from Japanese IBDV strains (Lin et al. (1993) "Avian Dis.", 37: 315-323) and Delaware variants A and E (Lana et al (1992) "Virus Genes", 6: 247-259; Heine et al. (1991) "J. Gen. Virol.", 22: 1835-1843). however nobody has complete the entire long segment of any United States IBDV variant Completely cloned and characterized.

epiphany the invention

The Inventors have now determined the area of the IBDV genome which for the antigenic variation is responsible. A DNA sequence containing the central variable region of the VP2 region, as well as a plasmid, containing this DNA sequence, Was constructed. This DNA sequence can be manipulated in this way that they desired virus neutralizing epitopes or immunogenic polypeptides of any IBDV strain. These immunogenic segments can turn be included in new recombinant IBDV vaccines.

Short description the drawing

1 illustrates the construction of various chimeric plasmids encoding IBDV-specific polyproteins. A map of the IBDV genome with its coding regions is shown at the top of the figure. Selected restriction sites are included in the figure: B, BamHI; E, BstEII; N, NdeI, R, NarI; S, SpeI. Dashed lines indicate the substitution of the D78 sequence (NdeI-NarI fragment) in the GLS sequence to restore the B69 epitope region. Solid lines and dotted lines indicate substitution of the E / Del-22 and DS326 sequences, respectively, in the GLS sequence to restore the B63 epitope region and to delete the 179 epitope region, respectively.

2 are electron micrographs of IBDV virus-like particles (| - | = 100 nm). A. Actually empty particles (without RNA) from purified virus. B. Virus-like particles (empty capsids) derived from a recombinant baculovirus that expresses the large genomic segment of IBDV in insect cells.

3 is a comparison of the deduced amino acid sequence of the structural proteins (VP2, VP3 and VP4) from ten IBDV strains. Dashed lines (-) indicate amino acid identity, and crosses (x) indicate a region where the sequence was not determined. Filled bars (|) indicate a gap in the sequence, and vertical arrowheads (↓) indicate the possible cleavage sites of VP2 / VP4 and VP4 / VP3. The two hydrophilic peaks in the variable region are overlined.

4 is a phylogenetic tree for the IBDV structural proteins using PAUP (phylogenetic analysis using Parsimony) Version 3.0 program (Illinois Natural History Survey, Champaign, Illinois).

5 represents the DNA and amino acid sequence for the GLS viral structural protein fragment VP2 / VP4 / VP3. A vertical line indicates the start / stop points for the VP2, VP4 and VP3 regions.

6 reflects the DNA and amino acid sequence for the E / Del22 virus structural protein fragment VP2 / VP4 / VP3.

IBD

– infektiöse Bursitis wie oben beschrieben.

IBDV-infektiöses

Bursitis-Virus, ein Virus, das in der Lage ist, wenigstens Läsionen in dem Schleimbeutel von Fabricius bei infiziertem Geflügel zu induzieren.

Epitopische Determinanten

– Aminosäuren oder Aminosäuresequenzen, welche Epitopen entsprechen, die durch einen oder mehrere monoklonale Antikörper erkannt werden. Die Gegenwart der Aminosäure oder Aminosäuresequenz an dem entsprechenden ORF-Ort verursacht, dass das Polypeptid das entsprechende Epitop aufzeigt. Eine epitopische Determinante wird durch Aminosäureidentität und Sequenzanordnung bestimmt.

Genetische epitopische Determinanten

– Nucleotidsequenzen des ORF, welche für epitopische Determinanten codieren.

Konformationale Epitope

– Epitope, die teilweise oder vollständig durch die quartäre (dreidimensionale) Struktur eines IBDV-Polypeptids induziert werden. Konformationale Epitope können das Binden zwischen einem IBDV und einem monoklonalen Antikörper verstärken oder können Bindung induzieren, wohingegen dieselbe Sequenz, der das konformationale Epitop fehlt, nicht in der Lage wäre, eine Bindung zwischen dem Antikörper und dem IBDV-Polypeptid überhaupt zu induzieren.

Virusartige Partikel

– dreidimensionale Partikel mit natürlichen oder rekombinanten Aminosäuresequenzen, die die dreidimensionale Struktur von IBDV nachahmen (für die das große Genomsegment A codiert), aber denen virale RNA fehlt. Virusartige Partikel zeigen konformationale Epitope auf, die durch native Viren mit ähnlicher Sequenz aufgezeigt werden. Virusähnliche Partikel werden für die angemessene Expression von DNA, die für VP2-, VP4-, VP3-strukturelle Proteine in einem angemessenen ORF codiert, entwickelt.

Epitopische Determinantenregion

– limitierter Bereich einer Aminosäuresequenz von VP2 von IBDV, die mit epitopischen Determinanten übersättigt ist, wobei die Variation unter Aminosäuren in diesem limitierten Bereich eine große Anzahl an Epitopen entstehen lässt, die durch unterschiedliche monoklonale Antikörper erkannt werden.

7 is a table of amino acid identities for essential sites (epitopic determinants) of eight different IBDVs.

IBD

Infectious bursitis as described above.

IBDV infectious

Bursitis virus, a virus capable of inducing at least lesions in the bursa of Fabricius in infected poultry.

Epitopic determinants

Amino acids or amino acid sequences corresponding to epitopes recognized by one or more monoclonal antibodies. The presence of the amino acid or amino acid sequence at the corresponding ORF site causes the polypeptide to display the corresponding epitope. An epitopic determinant is determined by amino acid identity and sequence ordering.

Genetic epitopic determinants

Nucleotide sequences of the ORF encoding epitopic determinants.

Conformational epitopes

Epitopes partially or completely induced by the quaternary (three-dimensional) structure of an IBDV polypeptide. Conformational epitopes can enhance binding between an IBDV and a monoclonal antibody or can induce binding, whereas the same sequence lacking the conformational epitope would be unable to induce binding between the antibody and the IBDV polypeptide at all.

Virus-like particles

- Three-dimensional particles with natural or recombinant amino acid sequences that mimic the three-dimensional structure of IBDV (encoded by the large genome segment A) but lack viral RNA. Virus-like particles display conformational epitopes indicated by native viruses of similar sequence. Virus-like particles are being developed for the appropriate expression of DNA encoding VP2, VP4, VP3 structural proteins in an appropriate ORF.

Epitopic determinant region

Limited range of an amino acid sequence of VP2 of IBDV supersaturated with epitopic determinants, the variation among amino acids in this limited region producing a large number of epitopes recognized by different monoclonal antibodies.

Best procedure to run the invention

Recombinant, immunogenic polypeptides that display the epitopes of two or more native IBDVs, as well as recombinant virus-like particles that display the epitopes of two or more native IBDVs, and conformational epitopes are effective immunogens for vaccines that can be used to confer protection against a wide variety of IBDV challenges in inoculated poultry. The recombinant polypeptides and virus-like particles are obtained by the expression of chimeric DNA produced by the insertion of epitopic determinants for at least a second IBDV into the VP2 region of basic IBDV. This is most easily accomplished by the substitution of the genetic epitopic determinants for the amino acid identities and arrangements found in 7 are shown. Therefore, where the epitopic determinant of the second IBDV differs from that of the basic IBDV, the epitopic genetic determinant for the different second IBDV is inserted in place of the genetic epitopic determinant at the site of the basic IBDV. An example of combining the epitopic determinants of D78, E / Del22 and DS326 IBDV into the basic GLS IBDV is in 1 executed. Thus, a DNA sequence can be made with genetic epitopic determinants for a variety of native IBDV. These recombinant plasmids can be used in a variety of Verpa including baculovirus, fowlpox virus, turkey herpesvirus, adenovirus and similar transfection vectors. The vectors can be used to infect conventional expression cells, such as SF9 cells, chicken embryo fibroblast cell lines, chicken embryo kidney cells, Vero cells and similar expression vehicles. Methods for transfection and methods for expression as well as plasmid insertion into transfection vehicles are well known and are not per se an aspect of the present invention.

The Expression of the chimeric cDNA of the invention produces immunogenic polypeptides, the epitopes of a variety from native IBDV, and expression of a recombinant VP2, VP4, VP3 cDNA segments, with the VP2 region, which in turn has genetic epitopic determinants for at least includes two native IBDV leaves immunogenic virus-like Particles are created.

The immunogenic polypeptides and virus-like Particles can be harvested using conventional techniques (Dobos et al. "J. Virol", 32: 593-605 (1979)). The polypeptides and virus-like Particles can used to make vaccines that provide protection for inoculated poultry be lent, especially chickens, and in a particularly preferred embodiment, broilers, protection against a challenge by every IBDV that contains an epitope, which in the multitude of epitopic determinants present in the inoculum are reflected. Thus, there is a single immunogen Occasion for immunity against a variety of IBDV, any IBDV, its genetic epitopic Determinant in the chimera cDNA was recorded.

The Administration of the vaccines can be effective according to well-established procedures accomplished become. U.S. Patent 5,064,646 discloses methods for the effective Inoculation of chickens described based on the then new isolation of GLS IBDV. Similar Administration and dosage frames may be used herein. Since the polypeptides and virus-like particles have no viral RNA, are they avirulent? The vaccines can therefore by the simple Involvement of immunogenic polypeptides and virus-like ones Particles in a pharmaceutical carrier, typically a suspension or mixture. Corresponding dosage values are best achieved through routine trial and error techniques found that the different antibody titers, which are induced, and / or the quantity of different epitopes, which are present, taken into account which is a complete crossimmunity to the to induce virulent challenge. In general, pharmacologically acceptable carriers, such as phosphate buffered saline, Cell culture medium, Marek's Virus vaccine diluent oil adjuvants and other auxiliaries, etc. may be used. The administration Preferably happens once to hens who are in the egg-laying season enter what provides the induction of an antibody, which is passed passively through the egg to the chick to an early one Prevent infection by virulent IBDV field strengths. The other way round Can the recombinant vaccine into a replication vector to each Time to be transmitted in the habitat of a chicken, preferably at the age of one day. Experience has shown that in the Generally, the level of protection is improved by a second inoculation can be.

The Invention can be further appreciated by reference to the specific examples, the running below are to be understood.

Examples

methodological background

Around to determine the molecular basis of antigenic variation in IBDV genomic segment A was of four IBDV strains, GLS, DS326, Delaware variant E (E / Del) and D78, cloned and characterized by sequencing. By comparing the deduced amino acid sequences of these strains with other serotypes 1- and Serotype 2 sequences previously published have been the putative amino acid residues identified in the binding with various neutralizing Mabs are involved, and the phylogenetic relationship of the IBDV structural proteins were examined.

GLS, DS326 and STC strains of IBDV were propagated in the bursa of specific pathogen-free chickens (SPA-FAS, Inc., Norwich, CT, USA). Tissue culture-adapted E / Del-22, D78 and OH (serotype 2) strains of IBDV were propagated and purified in primary chick embryo fibroblast cells derived from 10-day-old embryonated eggs (SPAFAS, Inc.) as described (Snyder et al. (1988a) "Avian Dis.", 32: 527-534). The Mabs against various IBDV strains were produced and characterized using protocols previously demonstrated (Snyder et al. (1988a) "Avian Dis., 32: 527-534, Snyder et al., 1988b, Avian Dis., 32: 535-539), Mabs B69 and R63 were prepared against a D78 strain, whereas Mabs 8, 10, 57, and 179 In addition, a new Mab 67 was prepared which was neutralizing and specific for the E / Del strain The identification of IBDV antigens by modified antigen capture ELISA (AC-ELISA) was as described (Snyder et al., (1992) "Arch. Virol.", 127: 89-101).

Various IBDV strains were due to their reactivities characterized by a variety of neutralizing Mabs, such as shown in Table 1.

TABLE 1 Antigen characterization of various IBDV strains after their activities with a selection of neutralizing MAbs

All Standard serotype 1 viruses reacted with Mabs B69, R63, 179 and 8, except PBG98 (a British vaccine strain, Intervet, UK), which is not reacted with Mab B69. In contrast, all US variants are missing Viruses the virus-neutralizing B69 epitope. In addition, GLS and DS326 variants are missing an epitope of R63, but they share a common epitope Mab57 is defined. Thus, these viruses were based on the reactivities with different mabs their antigenicity classified as classic, GLS, DS326 and E / Del variants.

Complementary DNA clones containing the entire coding region of the large RNA segment of various IBDV strains were prepared using standard cloning methods and methods previously described (Vakharia et al., (1992) Avian Dis., 36 : 736-742; Vakharia et al. (1993) J. Gen. Virol., 74: 1201-1206). The complete nucleotide sequence of these cDNA clones was determined by the dideoxy method using a Sequenaee DNA sequencing kit (US Biochem Corp., Columbus, OH). DNA sequences and deduced amino acid sequences were analyzed by a PC / GENE Software Package (Intelligenetics, Inc.). These are in the 5 and 6 shown. The nucleotide sequence data of the GLS strain was deposited with GenBank data libraries and assigned accession number M97346.

Comparisons of the nucleotide sequence of the GLS strain (3230 bp in length) with eight serotype 1 and one serotype 2 IBDV strain show a ≥ 92% and a ≥ 82% sequence homology, respectively; indicating that these viruses are closely related. It is interesting to find that there are only six to nine base substitutions between D78, PBG98 and Cu-1 strains, which corresponds to a difference of about 0.2% to 0.3% (results not shown). 3 and Table 2 show a comparison of the deduced amino acid sequences and the percent homology of the large segment A ORF of the ten IBDV strains, including four IBDV strains used in this study.

Table 2 Percent amino acid sequence homology of large segment A ORF from ten IBDV strains

These Comparisons show that the proteins are highly conserved. The degree the difference in the amino acid sequence ranked by 0.4% for the D78 - Cu-1 comparison and 10.3% for the Serotype 1 (E / Del) serotype 2 (OH) comparison (Table 2).

In 3 For example, sequence alignments of the deduced amino acid sequences of the large ORF (1012 residues) of ten IBDV strains (including the four used in this study) indicate that most amino acid changes occur in the central variable region between residues 213 and 332 of the VP2 protein as previously shown by Bayliss et al. (1990) J. Gen. Virol M., 71: 1303-1312. It is interesting to note that all US variants (GLS, DS326, and E / Del) differ from the other strains in the two hydrophilic regions that exist in 3 are swept over (residues 212 to 223 and residues 314 to 324). These two hydrophilic regions have been shown to be important in binding neutralizing Mabs and therefore may be involved in the formation of a virus neutralizing epitope (Heine et al., (1991) J. Gen. Virol., 22: 1835-1843). , Recently, we have shown that the conformationally-dependent Mabs B69, R63, 8, 179, 10 and 57 (see Table 2) immunoprecipitate VP2 protein (Snyder et al (1992) "Arch. Virol.", 127: 89-101). , In addition, E / Del-specific Mab 67 also binds to VP2 protein. Therefore, in order to identify the amino acids involved in the formation of virus-neutralizing epitopes, and therefore in the antigenic variation, we have compared the amino acid sequences of VP2 protein from classical and variant viruses.

Comparison of the D78 sequence with the PBG98 sequence reveals only four amino acid substitutions at positions 76, 249, 280 and 326. However, STC and 52/70 strains also differ from the D78 sequence at positions 76, 280 and 326 but these viruses bind to Mab B69. This implies that Gln at position 249 (Gln249) may be involved in binding with Mab B69. It should be noted that all US variant viruses have a Gln → Lys substitution at this position and therefore avoid binding with neutralizing Mab B69. Similarly, comparison of the GLS sequences with the DS326 sequence in the variable region shows six amino acid substitutions at positions 222, 253, 269, 274, 311 and 320. However, other IBDV strains that bind to Mab 179 have amino acid substitutions at positions 222, 253, 269, and 274, which are conservative in nature. Therefore, this suggests that Glu311 and Gln320 may be involved in binding with Mab 179. Again, a comparison of GLS and DS326 sequences with all other IBDV sequences shows a unique Ala → Glu substitution at position 321, suggesting the contribution of this residue to binding with Mab 57. Since Mab 57 does not compete with Mab R63, it is conceivable that Ala321 may contribute to binding to Mab R63. Similarly, comparison of the E / Del sequence with other sequences shows five unique substitutions at positions 213, 286, 309, 318 and 323. However, a comparison of this E / Del (tissue culture derived virus) sequence with previously published VP2-A / Del and -E / Del sequences (bursitis-derived virus) involved involvement of Ile286, Asp318 and Glu323 in binding to Mab67, as residues at positions 213 and 309 in the A / Del and E / Del sequences are not substituted (Heine et al., (1991) J. Gen. Virol., 22: 1835-1843; Lana et al. (1992) "Virus Genes", 6: 247-259 Vakharia et al. (1992) "Avian Dis.", 36: 736-742).

One Comparison of the amino acid sequence also shows a striking difference between serotype-1 and serotype 2 sequences. There is an insertion in the serotype 2 OH strain an amino acid residue at position 249 (serine) and a deletion of a residue at position 680. It has previously been shown that serotype 2 viruses are naturally avirulent are and no pathological lesions in chickens (Ismail et al. (1988) Avian Dis., 32: 757-759). Therefore, these could be minor changes in the structural proteins of the serotype 2-OH strain an important Role in pathogenicity play the virus. About that In addition, it was hypothesized that an amino acid sequence motif, S-W-S-A-S-G-S, (Residues 326 to 332) is conserved only in virulent strains and in the virulence could be involved (Heine et al. (1991) J. Med. Gene. Virol. ", 22: 1835-1843). This sequence motif was also present in different pathogenic strains of IBDV isolated in Japan (Lin et al. (1993) "Avian Dis.", 37: 315-323). One Comparison of Amino Acid Laws in this heptapeptide region, non-pathogenic serotype 2-OH strains are three Substitutions have, whereas mild-pathogenic strains of Serotype 1 (D78, Cu-1, PBG98 and 002-73) one or two substitutions in this area. About that In addition, a comparison of the hydrophilic plots of the variable region (Amino acids 213 to 332) of the variant serotype 1 strains and the serotype 2 OH strain a drastic reduction in the second hydrophilic peak area (Amino acid residues 314 to 324) for Serotype 2 (results not shown). Because most of the amino acid residues, which cause an antigenic variation to reside in this region, can these residues play an important role in the formation of virusneutralizing Epitopes as well as serotype specificity.

To evaluate the antigenic relationship of structural proteins of various IBDV strains, a phylogenetic tree was constructed based on the large ORF sequence of ten IBDV strains, including the US variant strains studied in this study ( 4 ), constructed. Three distinguishable stem lines were formed. The first, which is the furthest from the others, is the serotype 2-OH strain, and the second is the geographically distant Australian serotype 1 strain (002-73). The third lineage consists of four separate groups. The first and second groups include highly pathogenic strains, namely the standard challenge strain (STC) from the US and the British field strain (52/70). The third group comprises all European strains: the vaccine strains D78 (Holland), PBG98 (UK) and the mildly pathogenic strain Cu-1 (Germany). The fourth group consists of the US variant strains, in which E / Del forms a different subgroup. The groups formed by the phylogenetic analysis correlate well with the Mabs reactivity patterns (see Table 1). As in 4 all US variant viruses lacking the B69 epitope form a separate group, whereas all the classical viruses containing a B69 epitope constitute another group (except PBG98). In addition, the closely related GLS and DS326 strains containing a common Mab-57 epitope and lacking one R63 epitope could be separated from the other variant E / Del strain.

Based On this information was a recombinant vaccine as follows constructed:

Construction of recombinant Baculovirus clones containing chimeric IBDV genes

One recombinant baculovirus, which is a chimeric VP2, VP3 and VP4 structural Protein of the GLS strain was expressed, constructed and accepted. The recombinant baculovirus expressed a chimeric VP2 protein, including all Mab-defined GLS neutralization sites and a neutralization site (B69), which for classical strains of IBDV in the form of a VP2 VP $ VP3 segment.

Complementary DNA clones containing the entire coding region of the large RNA segment of GLS and D78 IBDV strains were prepared using standard cloning methods and methods previously described (Vakharia et al (1992) "Avian Dis. "36: 736-742; Vakharia et al. (1993)" J. Gen. Virol. "74: 1201-1206). To insert the gene sequence encoding the B69 epitope of the D78 IBDV strain, plasmid pB69GLS was constructed as follows (see 1 ). Full-length cDNA clones of D78 and GLS (plasmids pD78 and pGLS-5) were digested with NdeI-NarI and NarI-SpeI enzymes to generate an NdeI-NarI (0.26 kb) and a NarI-SpeI (respectively). 0.28 kb) fragment. These two fragments were then ligated into the NdeI SpeI cut plasmid pGLS-5 to obtain a chimeric plasmid pB69GLS. As a result of this insertion, three amino acids were substituted in the GLS VP2 protein. These substitutions were at positions 222 (Thr-Pro), 249 (Lys-Gln) and 254 (Ser-Gly) in the variable region of the VP2 protein (Vakharia et al (1992) "Avian Dis.", 36: 736 -742). In order to insert the chimeric IBDV structural genes into the baculovirus genome, plasmid pB69GLS was fully co-administered BstEII enzyme and partially digested with the BamHI enzyme, combined with the NheI-BstEII fragment (derived from the plasmid pGLSBacI, see Vakharia et al. (1993) "J.Gen.Virol", 74: 1201-120b) and then ligated into the NheI-BamHI cut transfer vector pBlueBacII (Invitrogen Corp., San Diego, CA). Finally, a recombinant baculovirus I-7 was obtained using prescribed procedures (Vakharia et al., (1993) J. Gen. Virol., 74: 1201-1206). See Table 3.

manufacturing an inoculum for the immunization

Spodoptera frugiperda SF9 cells infected with a multiplicity of 5 PFU per cell with the I-7 recombinant baculovirus, were used as suspension cultures in one-liter bottles, containing Hink's TNM-FH medium (HER Biosciences, Lenxa, KS) supplemented with 10% fetal calf serum, at 28 ° C for 3 to 4 Propagated days. The infected cells were removed by centrifugation recovered at low speed, washed twice with PBS and in a minimal volume of PBS resuspended. The cell slurry was on an ice bath three times for Sonicated for 1 min at 2 min intervals and by centrifugation clarified at low speed. An aliquot of each cell lysate was tested with anti-IBDV Mabs by AC-ELISA to the present to estimate antigenic mass (Snyder et al., 1998b) "Avian Dis., 32: 535-539) preparations with the largest antigenic Mass was pooled and comparatively in AC-ELISA against V-IBDV-7-1 recombinant baculovirus-IBDV vaccine titrated in a previous study (Vakharia et al., (1993) J. Med. Gene. Virol. ", 74: 1201-1206) has been used. The antigenic mass of the I-7 recombinant preparation, as determined by AC-ELISA with group-specific neutralizing Mab 8, was by dilution adjusted so that it was the same as in the V-IBDV-7-1 vaccine, and then she became an equal volume of Freund's incomplete adjuvant emulsified and for used the inoculation.

virus

The challenge viruses: The classical strains IM and STC and variant strains E / Del and GLS-5 were obtained from previously known sources (Snyder et al. (1988a) "Avian Dis.", 32: 527-534, Snyder et al. 1992) "Arch. Virol.", 127: 89-101). After intraocular instillation, the challenge viruses were titered into the bursae of specific pathogen-free (SPF) chickens (SPAFAS, Inc., Storrs, Conn.). For the strains STC, E / Del and GLS-5, a 100% chicken 50% infective dose (100 CID ₅₀ ) was determined based on the bursa-to-body weight measurements. One hundred lethal doses (100 LD) of the IM strain were calculated based on mortality 8 days post-inoculation (PI).

Hühnerinokulationen and IBDV challenge

White Leghorn SPF chicken were allowed to hatch in HEPA-filtered isolation units and reared (Monair Anderson, Peachtree City, GA). Eight weeks old chickens were pre-blotted, individually wing-cut, in 10 groups each with 15 chickens divided and treated as follows. Chickens of Groups I-V received no inoculations and served as either negative or positive Challenge controls. The chickens of the groups V-X were intramuscularly with 0.5 ml of the 1-7 inoculum, prepared above from recombinant baculovirus-infected cell lysates, inoculated. After 3 weeks of PI, all chickens were bled and the Chicken of Groups II-IX were with the corresponding IBDV challenge strain by ocular Instillation challenged. Four days after the challenge were 5 chickens each group humanely sacrificed and their kloakaler bursa became away. Each bursa has been processed and subsequently regarding the presence of IBDV antigen by AC-ELISA as described (Snyder et al. (1998b) "Avian Dis., 32: 535-539) Chicken classified as dead and humane in the IM challenged groups sacrificed when they were obviously doomed to death because of the IM challenge. Eight days after infection, the remaining chickens were in sacrificed and weighed to all groups. The bursa of Fabricius each chicken was carefully cut out and weighed as well. The Bursa-to-bodyweight ratio was for each Chicken, as described by Lucio and Hitchner (Lucio et al. (1979) "Avian Dis.", 23: 466-478). Each value for individually challenged chickens, by more than two standard deviation units from the mean the corresponding control group was used as a positive indicator for the Evaluated IBDV infection. Opened Bursae were buffered by immersion in 10% neutral buffer Formalin fixed. Transverse portions of bursae were processed by graded alcohols and xylene and embedded in paraffin, sectioned and with hematoxylinose colored and examined by a light microscope. Protection against the challenge was defined as the absence of any IBDV-induced lesions in the bursa of Fabricius.

serological rating

Of the classical D78 strain as well as the cell culture-adapted variant GLS strain of IBDV were in primary Chicken embryo fibroblast cells grown and used in virus neutralization (VN) tests, to serums from the vaccine trials essentially as described (Snyder et al. (1988a) "Avian Dis.", 32: 527-534) testing. Serum from the experiments was also under the presence of anti-IBDV antibodies Use of a commercially available IBDV antibody ELISA kit (Kirkegaard and Perry, Gaithersburg, MD).

rating of vaccines and challenge viruses

Of the antigenic content of the I-7 GLS chimera IBDV vaccine was checked in AC-ELISA with a selection of VP2 and VP3 specific Mabs. The relative antigenic mass of each epitope in the I-7 vaccine was expressed with previously tested batches of baculovirus compared to unmodified GLS subunit vaccines (Vakharia et al., (1993) J. Med. Gene. Virol. ", 74: 1201 to 1206). The status of each Mab-defined epitope on the I-7 chimeric vaccine was also compared to the status of those Mab-defined epitopes in wild-type IBDV Challenge Viruses that are used to increase the effectiveness of the I-7 vaccine to rate. Table 3 shows that antigenic mass levels in the 7-, 57- and B29 epitopes for the current one I-7 chimeric- Vaccine similar to a previously tested unmodified V-IBDV 7-1 GLS subunit vaccine were, but about four times higher as the original one unmodified V-IBDV-7 vaccine.

A major difference in the unmodified and chimeric vaccines was the appearance of the classic B69 epitope in the chimeric GLS product. The level of the B69 epitope was determined to be free as 9, as no comparison could be made with the unmodified GLS subunit vaccines. By comparing the status of Mab-defined epitopes on the challenge viruses with the unmodified and chimeric GLS subunit vaccines (Table 3), it could be found that while the chimeric product expressed the B69 epitope, it could be attributed to the classical STC and IMs. Challenge viruses was found, it also retained the entire homologous GLS epitopes.

Active cross guard

table 4 shows the results of a cross-protection test and serological tests Results obtained before the challenge.

Groups II-V served as challenge controls and, as shown by AC-ELISA, bursa-to-body weight and histological approaches, all unvaccinated chickens were fully susceptible to virulent IBDV challenge with all strains used. The IM challenge produced a lethal disease in one third of the chickens in the control group. in the In contrast, 8-week-old chickens, including VI-IX, were once vaccinated with the GLS chimeric vaccine, and for 3 weeks PI, all vaccinated chickens were fully protected against the challenge of all challenge viruses, including the lethal disease seen in controls by the IM strain was produced. Serological titres of reciprocal cross-VN assays performed with sera prior to challenge with D78 and GLS tissue culture virus were essentially two-fold different. The mean ELISA titers were relatively low, but they were also uniform between the vaccinated groups.

characterization the vaccines

In initial studies with baculovirus-expressed subunit GLS vaccines could the V-IBDV-7 GLS vaccines after administration of two doses (Table 3) only active antibody levels induce who were able to provide 79% protection against one homologous GLS challenge (Vakharia et al. (1993) J. Gen. Virol., 74: 1201-1206). In a subsequent study the antigenic mass of the original V-IBDV-7 vaccine became approximately fourfold elevated (calculated to the group specific Mab-8 location) and initiated One-dose and two-dose vaccination cross challenge trials with the unmodified GLS subunit vaccine designated V-IBDV-7-1 (Table 3). In these experiments, two doses of the vaccine resulted a complete one Cross protection against virulent STC, E / Del and GLS challenge. However, in the one-dose vaccine trial, while a complete Protection against the challenge with variant E / Del and GLS viruses was achieved, only 44% protection against the more distantly related classic Scored STC viruses. These studies could mean that simply by increasing the antigenic mass and / or the dose a better cross-protection could be obtained. However, it was just as obvious in the absence of homologous vaccines that low levels of antibodies by a dose of GLS V-IBDV 7-1 subunit vaccine, insufficient cross-protective against were a classic IBDV challenge. This could mean that at even lower levels of antibodies, as in cases of decaying maternal antibody, it would probably be that cross guard is reduced even more. In fact, even if not challenged with the STC virus, in some passive ones maternal antibody studies, performed using a different dosage of the V-IBDV-7 vaccine, while a homologous GLS protection guaranteed the progeny of vaccinated hens was only 57% against one more closely related E / Del challenge protected.

at Single dose vaccination cross challenge trials was the chimeric GLS I-7 vaccine, which contained the classic B69 neutralization epitope. To the present Attempt to make the previous attempts comparable the I-7 vaccine so formulated that by AC-ELISA the relative antigenic mass of the I-7 chimeric- Subunit vaccine approximate identical to the unmodified V-IBDV-7-1 vaccine, the previously used (Table 3). Table 4 shows the results the cross challenge after a single dose of the I-7 vaccine was administered. The results were similar to those associated with the unmodified V-IBDV-7-1 vaccine were obtained previously to the extent that protection against the GLS and E / Del strains was complete. However, the I-7 vaccine gave a complete one Protection against pathogenic and lethal challenge by the classical STC or IM strains. Since the antigenic mass of the GLS and the group epitopes on V-IBDV-7 and I-7 vaccines were well-equilibrated and equal it is reasonable close, that comparatively increase in the efficacy of the I-7 vaccine against the challenge with classic IBDV strains only and alone in the involvement of the classic IBDV-B69 neutralization epitope was due to the GLS VP2 protein sequence.

VIRUS SIMILAR PARTICLE

As noted above the recombinant cDNA and the immunogens of this invention expressed thereby attributed to the VP2 immunogenic region. In other words it may be sufficient to prepare a cDNA clone suitable for epitopic Determinants for a basic IBDV, e.g. GLS, as well as a second IBDV epitopic determinant, as D78, coded. Other epitopic determinants, all in the VP2 epitopic determinant region, may be included, cloned and expressed as described above.

As in 2 As shown, virus-like particles are formed by the expression of DNA encoding the VP2-VF4-VP3 structural protein sequences. These virus-like particle immunogens can be separated from the corresponding VP2-only immunogens, both with respect to the monoclonal antibody and by conventional separation techniques, such as electrophoresis and chromatography. The difference in reactivity with monoclonal antibodies, however, indicates that epitopes present in the VP2-VP4-VP3 structural protein sequence-induced virus-like particles are not present in immunogens expressed by the identical VP2 region only , These epitopes are "both linear and conformational" epitopes. Conformational epitopes are distinct from linear epitopes and are reflected in the conformation, not just the amino acid sequence of the actual virus. As a result, inoculation of poultry with a recombinant virus-like particle may even confer better protection against a field challenge of IBDV than inoculation with the immunogens of the VP2 region alone. This is due to the spontaneous accumulation of all structural elements of the virus.

Applicants have found that expression of the VP2 region as part of the VP2-VP4-VP3 structural protein single-segment forms virus-like particles such as those 2 , These particles have been shown to react with antibodies that do not react in the same manner with the identical recombinant VP2 immunogen. Therefore, virus-like particles can give rise to larger antibody titers and / or subtly different (broader) protection when inoculated with a poultry host.

The Invention herein therefore comprises (1) recombinant VP2 immunogens, comprising epitopic determinants of at least two different IBDV strains and (2) virus-like particles from VP2-VP4-VP3 segments, wherein the VP2 region again epitopic determinants of at least two different IBDV strains as well as the nucleotide sequences common to both 1 and 2, and vaccines comprising them.

Recombinant EPITOPE DETERMINANT COMBINATIONS

As in the over here executed Examples can be shown genetic epitopic determinants for an IBDV strain in the VP2 region a different basic IBDV genetic sequence are inserted and subsequently used to generate an immunogen-indicating epitope for both Express IBDV. Indeed the above examples demonstrate the combination of at least three different IBDV epitopic determinants. More can be combined become. The resulting vaccines contain an active agent the expressed immunogen, which provides protection against the challenge from a broad range of IBDV provides, rather than virus-based Vaccines of the prior art, which provide protection against a single Provide a tribe or a single family of tribes.

7 reflects the amino acid identities for the range of epitopic determinants for seven different IBDVs. These are not intended to be limiting, but they are representative. Desirable recombinant immunogens, both VP2 alone and virus-like particle VP2-VP4-VP3 immunogens, are obtained by substituting the genetic epitopic determinants for the varying amino acids at the identified sites in 7 (Arrays that are not identified are conserved among the IBDV strains). This induces the expression of the inventive immunogens. It is clear that the possible combinations are limited, even if they have a larger number, and that they can be examined routinely. Representative combinations will tend to reflect combinations of epitopic determinants of dominant IBDV.

A E / Del / GLS recombinant can change in the E / Del epitopic determinant region at position 213 Asn-Asp, 253 Gln-His and 269 Thr-Ser.

A DS326 / D78 recombinant may contain the amino acid and corresponding nucleotide substitutions on 76 Ser-Gly, 249 Lys-Gln, 253 Gln-His and 270 Ala-Thr substitutions:
Obviously, a wide variety of combinations is possible and will come to mind professionals. The epitopic determinant region, roughly including the region of amino acid 5-433 of the VP2 region, thus represents a recombinant "cassette" which can be tailored by site-directed mutagenesis to achieve amino acid insertion and / or deletion to desired recombinant To achieve cDNA clones, polypeptides, virus-like particles and vaccines with improved protection against a wide variety of IBDV.

LETALE IBDV, MONOCLONAL ANTIBODY and VACCINATE THEREOF

As noted, IBDV infection typically forms an immunosuppressive condition that is reflected in lesions in the bursa of Fabricius. This is typical of IBDV, which in the United States are encountered. However, there are lethal IBDV, that is, IBDV infections, which result in dying of chickens directly as a result of IBDV infection. While vaccines based on the isolation of these IBDVs have been developed, the resulting vaccines are "hot", that is, they self-induce or induce an immunosuppressive state, and the inoculated chicken must be provided with antibodies to other infectious agents. This method of protection is not desirable so that it will be discontinued in most commercial poultry houses in Europe. No adequate safe vaccine against lethal IBDV is currently available.

The Inventors have developed a monoclonal antibody, Mab 21, which under the Budapest Peace at the American Type Culture Collection, Accession. ATCC HB11566 was deposited. This monoclonal antibody is specific and for lethal IBDV strains neutralizing. The specificity is reproduced in Table 5, which confirms that, unlike others monoclonal antibodies, Mab 21 specific for is an epitope indicated only by IBDV strains with lethal potential becomes.

It should be noted that throughout this patent application, reference has been made to a variety of monoclonal antibodies used to confirm the presence of epitopes of different IBDV in the inventive recombinant chimeric immunogens of the application. These monoclonal antibodies were also deposited under the Budapest peace and are free he hältlich. However, they are not necessary to practice the invention and therefore they do not constitute an aspect of the invention. This should be contrasted with Mab 21.

As Other Mab developed by the inventors herein for IBDV may be a passive immunization against IBDV-lethal strains, in particular designed were standardized to immunization on a uniform Level and at any maternally derived levels against lethal IBDV field infection to reinforce to be obtained by vaccinating 1 day old chickens with a vaccine that comprises a pharmacologically acceptable carrier, such as those those described above in which an amount of Mab 21 is present which is effective in providing increased protection to inoculated chickens disposal to deliver.

The required level of protection can be achieved by a single dose of Vaccine administered in ova or to a 1 day old chicken, with a Mab-21 concentration of between 1 μg and 1 mg or by repeated Vaccinations with smaller effective doses that are carried out over time be lent. if repeated vaccinations should be used the dosage levels between 1 μg and 1 mg. The concentration level that is required to older chickens too vaccinate, increased with the weight of the bird and can be determined empirically.

A further investigation of the amino acid sequences of the lethal tribes in the epitopic determinant region reflects the highly conserved 279 identity Asn at position 279 of VP2 in non-lethal strains with a conserved Asp-identity at the same position in lethal trunks. Accordingly differentiates the epitopic determinant of the lethal tribe, which by Mab 21 is recognized and that is unique to the lethal strains, empirically of non-lethal IBDV but the substitution 279 Asp-Asn. According to the procedures outlined above, a chimeric recombinant Immunogen conferring effective protection against lethal IBDV, something that used to be with any type of vaccine without induction an immunosuppressive state was not possible by inserting the genetic epitopic determinant for 279 Asp in a non-lethal Basic IBDV, such as GLS. This will protect against the basic IBDV, the lethal IBDV, as well as all the other IBDV confer, whose genetic epitopic determinants are inserted. vaccines which are prepared from these immunogens, whether VP2 alone or in the form of virus-like particles from VP2-VP-VP3 segments used in the same way as mentioned above.

SEQUENCE LISTING

Claims (5)

A chimeric polypeptide immunogen comprising the VP-2 amino acid sequence of a first virus of infectious bursal disease (IBDV) or basic IBDV strain other than at least one amino acid X, where X is an epitopic determinant of a second IBDV strain, characterized that the polypeptide immunogen confers protection against classical IBDV or IBDV variant attack and stimulation and is selected from the group consisting of: a chimeric polypeptide immunogen, wherein the basic IBDV strain is GLS IBDV and the second IBDV strain D78 is IBDV and wherein the epitopic determinant from the second IBDV strain consists of three amino acid substitutions in GLS IBDV at positions 222, 249 and 254 of Thr by Pro, Lys by Gln and Ser by Gly; A chimeric polypeptide immunogen, wherein the basic IBDV strain is GLS IBDV and the second IB DV strain E / Del IBDV is and wherein the epitopic determinant from the second IBDV strain consists of five amino acid substitutions in the GLS IBDV at positions 269, 284, 286, 321 and 323 of Ser by Thr, Thr by Ala, Thr by Ile Gly is Asp, Glu is Ala and Asp is Glu; A chimeric polypeptide immunogen, wherein the basic IBDV strain is GLS IBDV and the second IBDV strain is DS326 IBDV and wherein the epitopic determinant from the second IBDV strain consists of four amino acid substitutions in the GLS IBDV at positions 269, 284 , 311 and 320 of Ser through Thr, Thr by Ala, Glu by Lys, and Gln by Leu; A chimeric polypeptide immunogen, wherein the basic IBDV strain is E / Del IBDV and the second IBDV strain is GLS IBDV and wherein the epitopic determinant from the second IBDV strain consists of three amino acid substitutions in the E / Del IBDV in the positions 213, 253, and 269 of Asn are Asp, Gln is His, and Thr is Ser; A chimeric polypeptide immunogen, wherein the basic IBDV strain DS326 is IBDV and the second IBDV strain D78 is IBDV and wherein the epitopic determinant from the second IBDV strain consists of four amino acid substitutions in the DS326 IBDV at positions 76, 249 , 253 and 270 of Ser through Gly, Lys through Gln, Gln through His and Ala through Thr. Vaccine conferring protection against IBDV in poultry, that a chimeric A polypeptide immunogen according to claim 1 and a pharmaceutically acceptable carrier includes. chimera cDNA which, when expressed as heterologous DNA into the DNA of an expression host functionally advertised, for the immunogen according to claim 1 coded. Transfection vehicle for the infection of an expression host, comprising the cDNA according to claim 3, as being functional with the Baculovirus fowlpox virus DNA, Turkey herpesvirus or adenovirus. Expression vehicle for the expression of the immunogen according to claim 1 comprising an expression host selected from the group consisting of SF9 cells, chick embryo fibroblast cells, Chicken embryo kidney cells and Verozellen transfected with the vehicle according to claim 4 are, selected is.